The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2505-2511

High Maternal Intake of Polyunsaturated Fatty Acids During Pregnancy in Mice Alters Offsprings' Aggressive Behavior, Immobility in the Swim Test, Locomotor Activity and Brain Protein Kinase C Activity^1,2,3

Margarita Raygada^*, , Elizabeth Cho^{*, **}, and Leena Hilakivi-Clarke^{*, , 4}

^* Lombardi Cancer Center,  Department of Psychiatry and ^** Department of Physiology and Biophysics, Georgetown University, Washington, D.C. 20007

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Populations in Western countries consume an excess of polyunsaturated fatty acids (PUFA), even during pregnancy. Since (n-6) PUFA is critical for brain development, we studied whether a high maternal consumption of this fatty acid alters offsprings' affective-like behaviors and (n-6) PUFA-induced protein kinase C (PKC) activity in the brain. Three different strains of pregnant mice were fed isocaloric diets containing either 16% (control) or 43% (high) energy derived from fat high in (n-6) PUFA (corn oil: Balb/c and CD-1 mice, or soybean oil: C3H mice) throughout gestation. From birth onward dams and offspring were fed a nonpurified diet containing 12% energy from a variety of fats. Two- to 12-month-old female and male offspring of dams exposed to a high (n-6) PUFA diet during pregnancy were significantly more active in an open field, more aggressive in the resident-intruder test and spent less time immobile in the swim test than offspring of dams exposed to a control (n-6) PUFA diet. Significantly greater PKC activity in the hypothalamus and moderately less PKC activity in the whole brain (P = 0.10) were seen in the 2-month-old female and male high (n-6) PUFA offspring compared to controls. Our findings indicate that in utero exposure to a high (n-6) PUFA diet subsequently increases locomotor activity and aggression, and reduces immobility in the swim test. The mechanism mediating these effects may be linked to an increased PKC activity in the hypothalamus.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The long-chain (n-3) and (n-6) polyunsaturated fatty acids (PUFA)5 are important constituents of cell membranes, particularly in the central nervous system (CNS). They are derived from essential fatty acids, -linolenic acid and linoleic acid, respectively, that can only be obtained from diet. Therefore, an inadequate maternal intake of the long-chain (n-3) and (n-6) PUFA leads to abnormal brain development (e.g., Connor et al. 1992, Wainwright 1992), including changes in visual functions (Neuringer et al. 1986, Uauy et al. 1992) and possibly in memory and behavior (Lamptey and Walker 1976). A high dietary intake of PUFA also may be detrimental, as indicated by impaired learning in rats fed a diet high in (n-6) fatty acids (Yamamoto et al. 1987) and lower scores of psychomotor development in infants fed with formulas supplemented with (n-3) fatty acids (Carlson et al. 1994).

Many people in Western countries consume an excess of food. Even the most educated pregnant women consume more fat and fewer carbohydrates than the international dietary recommendations suggest (Alberti-Fidanza et al. 1995). Therefore, it is important to determine the effects of a high PUFA diet on different behaviors. Our data in adult male mice and rats indicate that a high (n-6) PUFA diet increases aggressive behavior (Hilakivi-Clarke et al. 1996a). The present study investigated whether a maternal intake of a high (n-6) PUFA diet during pregnancy also alters aggressive behavior in the offspring. The other behaviors studied were locomotor activity and immobility in the swim test.

We also examined some of the possible mediators of high in utero dietary (n-6) PUFA exposures on behavior. In our preliminary analysis (performed by Dr. Norman Salem at National Institutes of Alcohol Abuse and Alcoholism, Bethesda, MD), the brain fatty acid composition of 3- or 12-month-old mouse offspring exposed to a diet containing either 16% or 43% energy from fat through their pregnant mother did not reveal any marked differences. The fat sources were either corn oil or soybean oil, both of which contain high levels of (n-6) PUFA. Because no differences were seen, the brain lipid composition was not studied here. Instead, we investigated whether protein kinase C (PKC) activity in the brain is altered. The PKC family of genes is part of a signal transduction pathway that is activated by hormones (i.e., estrogens), cytokines and growth factors (Nishizuka 1992). PKCs also are dependent on diacylglycerol (a fatty acid metabolite) and calcium for activation (Dekker and Parker 1994). This enzyme is highly localized in the brain (Saito et al. 1988) and appears to play a crucial role in regulating neurotransmission (Nishizuka 1992). There are several reasons to expect that a high maternal (n-6) PUFA diet may alter brain PKC activity in the offspring. First, a diet high in (n-6) PUFA increases circulating estrogen levels (Adlercreutz 1991), including during pregnancy (Hilakivi-Clarke et al. 1996b and 1997b). Estrogens, in turn, increase expression of some PKC isoforms in the uterus (Cutler et al. 1994) and perhaps in the brain (Ansonoff and Ametgen 1996). Second, a high-fat diet is shown to elevate PKC activity, at least in the mammary gland (Hilakivi-Clarke et al. 1998), colon (Reddy et al. 1996) and epidermal cells of the skin (Choe et al. 1992). Third, affective behaviors, including bipolar depression and alcohol-related behaviors, may be partly regulated by PKC (Manji et al. 1993).

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets

Seven-week-old female and male mice were kept on AIN93-based diets (Reeves et al. 1993) containing either 16% energy from fat [control (n-6) PUFA diet] or 43% energy from fat [high (n-6) PUFA diet]. When the mice had been fed these diets for 1 wk, females were mated. We had approximately eight pregnant mice per group that gave birth to litters containing six to 13 offspring. The pups were kept with their biological mother until weaning on postnatal d 25. In all studies done with the Balb/c and CD-1 mice, the fat source was corn oil, and in the studies done with the C3H/OuJ mice, the fat source was soybean oil. Both oils are high in (n-6) PUFA linoleic acid (corn oil: 59% linoleic acid of total fatty acids; soybean oil: 56%). The requirement of diet-derived linoleic acid during pregnancy in rodents is 4.5% (~0.4 g/d) of energy (Holman et al. 1991). In our study, a mouse fed a control (n-6) PUFA diet during pregnancy obtained ~0.8 g/d linoleic acid, and a mouse fed a high (n-6) PUFA diet obtained 2.3 g/d linoleic acid. Soybean oil also contains high levels of -linolenic acid (8%), while the level of this (n-3) PUFA in corn oil is enough to fulfill the nutritional requirements during pregnancy (1%).

The semipurified AIN93 (American Institute for Nutrition 1993) animal diets were prepared by Bioserv (Frenchtown, NJ) (Table 1). Although the high and control (n-6) PUFA diets had significantly different fat contents, the diets were isocaloric due to adjustments made using fiber (alphacel). Thus, the high (n-6) PUFA diet contained more fiber than the control diet. Isocaloric diets were used instead of nonisocaloric diets, since if fed a nonisocaloric high (n-6) PUFA/high energy diet, pregnant rodents reduce their feed and energy intake below that of rodents fed low (n-6) PUFA/low energy-density diet (Guo and Jen 1995). The proportion of energy of dietary components other than fat were adjusted to ensure an adequate intake of protein, carbohydrates, vitamins and trace elements, and the amounts of these components per diet were approximately constant with regard to energy. The pregnant mice consumed the high or control (n-6) PUFA fat diets until the delivery of the offspring. On the day the offspring were born, the diets were switched to a nonpurified diet (Purina Rodent Laboratory Chow 5001) that contains 12% energy from fat (saturated and unsaturated). The physiological energy value of this diet is 3.3 kcal/g (13.8 kJ/g).

Behavioral testing procedures

Locomotor activity.  The test apparatus was an open rectangular box (30 × 40 × 15 cm) made of transparent Plexiglas. The floor of the open box was divided into 12 identical squares. Each mouse was put individually into the lower right corner of the box, and the number of entries into the squares was scored during the 3-min observation period. The box was cleaned after each test.

Swim test.  Immobility in the swim test was used to measure depressive-like behaviors in mice. This test predicts the antidepressant efficacy of different drugs (Porsolt et al. 1977) and the effects of a variety of stressors on behavior (Garcia-Marquez and Armario 1987). In general, antidepressants shorten the time spent immobile in the water, whereas stressors lengthen it. Further, neonatal clomipramine treatment lengthens immobility in the swim test and induces subsequent behavioral changes which closely resemble those seen in clinical depression (Vogel et al. 1990). Thus, the swim test may be an index of depressive-like behaviors (Weiss et al. 1981). In the swim test, a mouse was placed in a plastic cylinder (height 17 cm, diameter 21 cm) containing 8 cm of water maintained at ~25°C for 8 min. This 8-min period included a 2-min acclimation period at the beginning of the test, immediately followed by a 6-min test period (Hilakivi et al. 1989a). The time spent immobile in the water was scored using a stop watch. A mouse was judged to be immobile when it was floating almost motionless.

Resident-intruder test of aggression.  The mice were isolated for 7 d. They were then confronted in their home cage with a group-housed, same-sex intruder which had the same in utero exposure but that had no previous contact with the resident. Our logic was to have intruders which would have behaved in a similar manner to the residents, if they had been housed in isolation. Another approach would have been to use intruders whose mothers were fed with Purina lab chow during pregnancy to standardize intruders' behavior. During a 5-min test period, the duration of social investigation (sniffing, following, grooming) and aggression (lateral threats, a tail rattle, biting, fighting) (Hilakivi and Lister 1989, Miczek 1987) was recorded with stop watches.

Brain weights

PKC activity

PKC activity was determined using the Biotrak PKC enzyme assay system obtained from Amersham Life Sciences (Arlington Heights, IL) following the manufacturer's instructions. On this assay, the level of PKC activity was determined using a specific peptide derived from the epidermal growth factor receptor. All reactions were set up on ice. Brain samples were homogenized in a buffer containing 50 nmol/L Tris/HCl pH 7.5, 3 g/L -mercaptoethanol, 5 mmol/L EDTA, 10 mmol/L EGTA and 50 mg/L phenylmethylsulphonylfuoride. Blanks were used to correct for nonspecific effects of [³²P] ATP or the binding of its radiolytic decomposition products. Extracts of MDA-MB-231 human breast cancer cell line which contain high PKC activity were used as positive control.

Statistical analysis

	RESULTS

Abstract Introduction Methods Results Discussion References

Maternal indexes and early development of the offspring

Behavioral patterns

Locomotor activity.  In utero exposure to a high (n-6) PUFA diet did not significantly alter locomotor activity during a 3 min test in an open field in female C3H mice (males not studied) or female and male Balb/c mice, when compared with an exposure to a control (n-6) PUFA diet in utero (Fig. 1). Among the CD-1 mice, however, offspring of dams exposed to a high (n-6) PUFA diet during pregnancy exhibited higher locomotor activity scores than offspring of control (n-6) PUFA dams among female (t = 4.6, df = 17, P < 0.0003) and male (t = 3.1, df = 18, P < 0.006) mice (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig 1. Effects of fetal exposure to a high (43% of energy from fat) or control (16% of energy from fat) (n-6) polyunsaturated fatty acid (PUFA) diet on locomotor activity in the 3-min open field test among 12-mo-old C3H mice (fat source soybean oil), 3-mo-old Balb/c mice (corn oil) and 2-mo-old CD-1 mice (corn oil). Values are means ± SEM, n = 8-10 mice per group. *Significantly different from the control (n-6) PUFA offspring, P < 0.05.

Swim test.  Time spent immobile in the swim test was significantly shorter in the female offspring of the C3H (t = 2.8, df = 17, P < 0.01), Balb/c (t = 2.4, df = 10, P < 0.04) and CD-1 mice exposed to a high (n-6) PUFA diet (t = 4.7, df = 31, P < 0.0001) than in the control (n-6) PUFA offspring (Fig. 2). Similarly, the male Balb/c (t = 3.1, df = 18, P < 0.006) and CD-1 offspring (t = 2.9, df = 18, P < 0.01) of mothers fed a high (n-6) PUFA diet during pregnancy spent a shorter time immobile than the respective control mice (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig 2. Effects of fetal exposure to a high (43% of energy from fat) or control (16% of energy from fat) (n-6) polyunsaturated fatty acid (PUFA) diet on immobility in the water in the 6-min swim test among 12-mo-old C3H mice (fat source soybean oil), 3-mo-old Balb/c mice (corn oil) and 2-mo-old CD-1 mice (corn oil). Values are means ± SEM, n = 10-17 mice per group. *Significantly different from the control (n-6) PUFA offspring, P < 0.05.

Resident-intruder test of aggression.  Aggressive behavior in the resident-intruder test was higher in the female C3H (t = 2.9, df = 16, P < 0.01) and CD-1 offspring (t = 2.8, df = 17, P < 0.01) of the high (n-6) PUFA group than of the control (n-6) PUFA group (Fig. 3). Among the Balb/c mice, four of eight female mice (50%) exposed to a high (n-6) PUFA diet in utero exhibited aggression, while none of the controls exhibited aggression in the resident-intruder test. Thus, the female Balb/c offspring of high (n-6) PUFA dams were more aggressive than the female offspring of control (n-6) PUFA dams (² = 5.3, df = 1, P < 0.025) (Fig. 3). The male CD-1 mice exposed to a high (n-6) PUFA diet in utero exhibited significantly higher levels of aggressiveness than the male mice exposed to a control (n-6) PUFA diet in utero (t = 5.4, df = 16, P < 0.0001) (Fig. 3). The level of aggression among the male Balb/c mice was low, as we have also reported earlier (Hilakivi and Lister 1989), and no significant differences were seen between the offspring of dams exposed to a control and high (n-6) PUFA diet (Fig. 3).

View larger version (13K): [in this window] [in a new window]   Fig 3. Effects of fetal exposure to a high (43% of energy from fat) or control (16% of energy from fat) (n-6) polyunsaturated fatty acid (PUFA) diet on aggressive behavior in the 5-min resident-intruder paradigm among 12-mo-old C3H mice (fat source soybean oil), 3-mo-old Balb/c mice (corn oil) and 2-mo-old CD-1 mice (corn oil). Values are means ± SEM, n = 8-10 mice per group. *Significantly different from the control (n-6) PUFA offspring, P < 0.05.

Brain weight

PKC activity

View larger version (19K): [in this window] [in a new window]   Fig 4. Effect of in utero exposure to a high (43% of energy from corn oil) or control (16% of energy from corn oil) (n-6) polyunsaturated fatty acid (PUFA) diet on protein kinase C (PKC) activity in the total brain, hypothalamus and frontal cortex in 2-mo-old female and male Balb/c mice. Each bar represents mean ± SEM, n = 5-8. *P < 0.05.

In accordance with the data obtained in female mice, the PKC activity was significantly higher in the hypothalamus (P < 0.05) and tended to be lower in the whole brain of male mice exposed to a high (n-6) PUFA diet in utero (F(1,14) = 4.20, P < 0.06) (Fig. 4). The PKC activity in the males also was significantly higher in the whole brain than in the hypothalamus (F(1,14) = 185.26, P < 0.0001). This was not the case in the female mice.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

A maternal exposure to high levels of (n-6) PUFA during pregnancy alters some affective-like behaviors in the offspring. The original observation that a high maternal (n-6) PUFA intake during pregnancy alters offsprings' behavior was obtained using 12-mo-old female C3H mice. However, we felt that we had to repeat these studies using two other mouse strains, inbred Balb/c mice and outbred CD-1 mice, for the following three reasons. 1) C3H mice carry a MMTV-mediated virus and may therefore develop spontaneous mammary tumors at 12-16 mo. We were concerned that the data may change if other mouse strains are used. 2) In utero manipulations may cause notable physiological alterations only in relatively old individuals (Vom Saal et al. 1997). Thus, we studied the effects of high fetal (n-6) PUFA levels on behavior in 2-3 mo-old mice. 3) Soybean oil was used in the C3H mice, and it contains relatively high levels of (n-3) PUFA as well as (n-6) PUFA. However, humans on a high-fat diet are likely to consume diets that are high in (n-6) PUFAs but not (n-3) PUFA. We therefore used corn oil as a source of maternal fat in studies performed using Balb/c or CD-1 mice.

Both male and female offspring, of at least one strain of mice, exposed to a high (n-6) PUFA diet through a pregnant dam exhibited shortened immobility in the swim test, greater aggressiveness in the resident-intruder paradigm and greater locomotor activity in an open field. Similar effects were noted in the corn oil and soybean oil groups. Since soybean oil contains high levels of both (n-6) linoleic acid and (n-3) fatty acids, and corn oil contains high levels of linoleic acid and sufficient levels of -linolenic acid, the behavioral alterations in the offspring appear to be mostly due to a high dietary (n-6) fatty acid content. In addition, different strains of animals often differ in their response to various manipulations (Belknap et al. 1998, Crawley et al. 1997), as also observed here. The locomotor stimulation by a high fetal (n-6) PUFA exposure was seen only in the CD-1 mice but not in the Balb/c or C3H mice. Female mice of all three strains exhibited greater aggressiveness when exposed to a high (n-6) PUFA diet in utero; however, increased aggressiveness was seen only in the male CD-1 mice. Thus, the outbred CD-1 strain appears to be more sensitive to the effects of a high maternal (n-6) PUFA diet than the inbred Balb/c or C3H mice.

PKC activity can be influenced by a high (n-6) PUFA diet. (n-6) PUFA increases the activity of this enzyme in several target organs, including the colon (Choe et al. 1992), mammary gland (Hilakivi-Clarke et al. 1998) and skin (Reddy et al. 1996). In the present study, the hypothalamic PKC activity was significantly increased in the offspring of high (n-6) PUFA-fed dams, and a similar trend was seen in the frontal cortex. However, since the whole brain PKC activity was slightly lower in the high (n-6) PUFA offspring than in the control (n-6) PUFA offspring, it is likely that, in contrast to the hypothalamus and frontal cortex, some areas of the brain of offspring of high (n-6) PUFA dams exhibit significantly reduced PKC activity. Our earlier results indicate that a maternal exposure to a high (n-6) PUFA diet during pregnancy reduces PKC activity in the mammary gland (Hilakivi-Clarke et al. 1998). This finding and the tendency for a lowered PKC activity in the whole brain assays suggest that a high maternal (n-6) PUFA diet during pregnancy may cause a permanent down-regulation of PKC in the offspring in certain tissues. In the hypothalamus, however, PKC activity appears to be permanently elevated.

The increases and decreases in PKC activity in the brains of offspring of high (n-6) PUFA dams may indicate that different isoforms of PKC are influenced in different parts of the brain by the high fetal (n-6) PUFA exposure. This interpretation is supported by the observation that expression of various PKC isoenzymes vary in the brain and some isoforms are particularly high in some brain areas and low in others (Saito et al. 1988). In addition, PKC isoenzymes in different brain areas may respond differently to manipulations (Komachi et al. 1994). Although PKC may participate in the regulation of certain brain functions, including memory, manic-depressive illness and alcohol-related behaviors (Manji et al. 1993, Serrano et al. 1995), it is not known whether PKC is linked to aggression or locomotor activity. The results of the present study suggest that the increased hypothalamic PKC activity may be associated with increased aggressiveness, shortened immobility in the swim test and increased locomotor activity. It is, thus, possible that PKC in the brain is the biological mediator of a high maternal (n-6) PUFA diet on offsprings' behavior.

Another mechanism of action of a high maternal (n-6) PUFA diet on offsprings' behavior could be through increased circulating estradiol levels in pregnant dams (Hilakivi-Clarke et al. 1996b and 1997b). There is compelling evidence to suggest that gonadal hormones in utero and during the first few postnatal days exert permanent organizational effects on several behaviors (Beatty 1992). For example, an exposure to estradiol or the synthetic estrogen diethylstilbestrol (DES) immediately after birth increases subsequent potential for aggressive behavior in female and male mice (Hilakivi-Clarke et al. 1997a, Simon and Whalen 1987a). Further, male knockout mice without a functional estrogen receptor  are not aggressive (Korach 1994). The increased aggressive encounters between the female and male offspring of dams consuming a high (n-6) PUFA diet during pregnancy and exhibiting high pregnancy estrogen levels (Hilakivi-Clarke et al. 1996b and 1997b) supports the view that an excess of (n-6) PUFA in utero may increase aggressive behavior by elevating mothers' and fetus' estrogen levels. A high (n-6) PUFA diet increases circulating estrogens only at the time of the exposure to this diet, i.e., during pregnancy. Thus, the offspring probably have high estradiol levels in utero, whereas after birth no differences in circulating estrogens can be detected (Hilakivi-Clarke et al. 1997b).

High in utero levels of estrogens have been linked to lengthened, not shortened, immobility in the swim test. Male mice treated with estradiol during postnatal d 1, 2 and 3 (the period during which the circulating estrogens originating from the mother/placenta are still high and the offspring are sensitive to maternal estrogens) spend a longer time immobile in the water than vehicle-treated controls (Hilakivi-Clarke et al. 1997a). Human studies also indicate that an exposure to DES in utero may increase depressive tendencies (Pillard et al. 1993). In contrast, treatment with estradiol immediately after birth does not affect behavior in the swim test in female mice (Hilakivi-Clarke et al. 1997a). The data in the present study, that show a shortened immobility in the swim test in the male and female high (n-6) PUFA offspring, suggest that other mechanisms besides an elevation in circulating estradiol are responsible of this behavioral change. The increase in locomotor activity in the female and male CD-1 mice exposed to high (n-6) PUFA in utero also contradicts the idea that a high maternal (n-6) PUFA diet affects offspring through a change in fetal estrogenic environment. Fetal estrogens are not thought to be important for locomotor activity (Beatty 1992).

In summary, we found that a maternal exposure to a high (n-6) PUFA diet during pregnancy increases aggressiveness and locomotor activity, and shortens immobility in the swim test in male and female mice offspring. The results further show that a high maternal (n-6) PUFA exposure increases offsprings' PKC activity in the hypothalamus. It remains to be determined whether hypothalamic PKC activity is causing the behavioral changes. These data may explain some of the proposed associations between behavior and risk of a certain disease. For example, increased breast cancer risk is linked to various personality characteristics, including anti-emotionality (Bleiker et al. 1996). A maternal exposure to a high (n-6) PUFA diet during pregnancy increases the incidence of mammary tumors in the offspring (Hilakivi-Clarke et al. 1997b, Walker 1990b) and may do so among humans (Michels et al. 1996). Since high (n-6) PUFA offspring also are "anti-emotional," as indicated by shortened immobility in the swim test, behavior and cancer risk may share a common biological basis, i.e., a high maternal intake of (n-6) PUFA during pregnancy.

	FOOTNOTES

Manuscript received 27 January 1998. Initial reviews completed 18 March 1998. Revision accepted 11 August 1998.

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